The primary transcripts of eukaryotic structural gene (precursor mRNAs; pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing. In some instances, alternative splicing of a common pre-mRNA provides an important mechanism for regulating gene expression. During the previous period of funding we have made significant progress in several areas relating to constitutive and regulated splicing. During the next budget period we will expand upon these successful programs. A major accomplishment was to purify, clone and characterize U2 snRNP Auxiliary Factor (U2AF) an essential splicing factor that initiates spliceosome assembly. U2AF contains an arginine-serine rich (RS) motif, present in a variety of higher eukaryotic splicing factors and regulators. We have shown that the RS region of U2AF is a splicing "effector" domain. A major priority will be to understand the mechanism by which the RS domain functions to promote binding of U2 snRNP to the branch point and hence spliceosome assembly and splicing. This is part of a more long-term goal to understand early events in splicing complex assembly. A major unanswered question is what components of the spliceosome participate in catalysis? We will perform photocrosslinking experiments to gain insight into this long-range question as well as aspects of early splicing complex formation. A major goal in the splicing field is to understand how alternative splicing is regulated. Over the past budget period we have developed an in vitro system that supports a splice-site switch following addition of the Drosophila Sxl protein, a known splicing regulator. Over the next budget period we propose a series of experiments to study this regulated splicing event. Relevant to this goal are experiments to understand the basis by which two polypyrimidine tract binding proteins, U2AF and Sxl, have overlapping but non-identical RNA binding specificities. Following splicing, the mature mRNA is exported from the nucleus to the cytoplasm. The mechanisms involved in this fundamental process remain largely unknown. We have made a series of observations suggesting the potential involvement of a glycolytic enzyme, GAPDH, in RNA export. Experiments are proposed to investigate this possibility.